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Gene targeting of -mediated signaling molecule, Cas-L/HEF1/NEDD9 reduced the None severity of collagen-induced arthritis.

Possible involvement of Cas-L in the pathophysiology of RA.

Satoshi Iwata, Tomoki Katayose, Yoshiko Kichikawa, Hiromi Ichihara, Hiroshi Kawasaki, Osamu Hosono, Hirotoshi Tanaka and Chikao Morimoto Institute of Medical Science, The University of Tokyo

Integrins:

1. out 2. Cas (Crk-associated substrate) family in co-incidence of a/b heterodimer (a chain 17, b chain 8, 22 ） 3. HTLV-I tax Tg mice and Cas-L: Non-covalent association VLA (very late antigen): b1 integrin arthritis and Cas-L induction, cell infiltration and Cas-L Ligand: ECM (extracellular matrices) and cell surface molecules expression a1b1 (VLA-1) Col, LN-1 a2b1 (VLA-2) Col, LN-1 a3b1 (VLA-3) FN, Col, LN-5 4. Collagen-induced arthritis (CIA) on Cas-L -/- a4b1 (VLA-4) FN (EILDV), VCAM-1, MAdCAM-1, TSP a5b1 (VLA-5) FN (RGD) mice: severity, incidence, histology, cytokine and a6b1 (VLA-6) LN-5 a7b1 LN-1 autoantibody levels a8b1 FN, VN, TNC a9b1 TNC avb1 VN

1980s： Development of mAb (Morimoto) 1992: cDNA of NEDD9 Integrins/ 4B4→integrin b1; neural precursor cell expressed, 3G6, 8F2→integrin a4; developmentally down-regulated 9 Actin-binding proteins (ABPs): 2H6→integrin a5 2C3A→integrin a6 1991： In epithelial cells Vinculin, Talin, Tensin, a-actinin b1 integrin induces tyrosine

Protein tyrosine kinases (PTKs): 1990s： 1992： cDNA cloning of FAK FAK (focal adhesion kinase) Co-stimulation (Matsuyama) /Pyk2 (proline-rich tyrosine kinase) 1993: FAK in T cells and B cells Src family (Src, Fyn) Tyrosine-phosphorylated pp105 (Nojima) 1994: cDNA cloning of p130Cas MAPK system: v-Crk or v-Src transformed cells (3Y1)

Grb2-Sos-Ras-Raf-MEK-Erk 1995: Binding of FAK and paxillin (pp70) 1995: cDNA and pp130 (Tachibana) cloning of Efs Small GTPases: Rac, Rho, cdc42, Ras (Sasaki) 1996：cDNA cloning of Cas-L (pp105) 1996: cDNA cloning 1996: cDNA cloning Adaptor proteins: Crk, Nck, Paxillin, (Tachibana) of HEF1 of Sin (Golemis) (Baltimore) Docking proteins: Cas 1997:FAK and Src family PTK (Crk-associated substrate) tyrosine-phosphorylate Cas-L (Tachibana)

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Structure and functions of Cas family proteins Rheumatoid Arthritis CTLA-4 TLR CD28 CD80/86 VCAM-1↑FN↑ Lumen Abl Crk Nck Src APCs Th0 SHP-2 Csk Dim1p Ⅱ TCR IL-2 N-terminal C-terminal MHC YDYVHL CD40L macrophages IFNγ CD4 β1 integrins↑ Nedd9 Th1 CD40L Cas-L SH3 SD (YXXP)13 SR CC HLH CT IL-8 FcR CD28 HEF1 834aa G-CSF Th17 GM-CSF Cas-L p130Cas FAK EGF-R CD40 Id2 Smads TNF-α PYK2 CD80/86 B cells PTP-PEST IL-1 monocytes YDYVHL IL-17 CD20 Neutrophils CD22 BCAR1 SH3 PP SD (YXXP)15 SR PPCC HLH CT IL-6 p130Cas HUVECs YDYVHL 874aa CC ,CXC ChondrocytesCartilage destruction HLH RANK Efs MMPs Auto-antibodies （RF, CCP Ab SH3 PP SD (YXXP)8 SR PP PP 560aa Sin RANKL Anti-typeⅡ collagen Ab） Bone destruction CC CT OC Synovial fibroblast (Iain B. Mclmmes, et al, N Engl J Med,2011)

HTLV-I tax Tg mice: RA model mice Histology (HTLV tax Tg mice)

HTLV-I genome

env pX

WT wRA

LTR gag pol tax LTR Tg

rex 21bp enhancer

Tg wRA

w/o RA Tg

Tg wRA

w RA Tg

(Miyake-Nishijima et al, Arthritis Rheum.,2003) (Miyake-Nishijima et al, Arthritis Rheum.,2003)

Human RA: Cas-L -/- mice: Seo S and Hirai H et al. J. Immunol. 2005 Accumulation of Cas-L+ T cells

100μm 100μm

100μm 100μm

Miyake-Nishijima et al, 100μm 100μm Arthritis Rheum.,2003

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Type II collagen-induced arthritis (CIA) CIA scoring and incidence Preparation of emulsion ① type Ⅱ chicken collagen（2 mg/ml in First immunization Second immunization 10mM acetic acid)：3 ml ②FCA (Freund’s complete adjuvant) 3 ml + Mycobacterium tuberculosis 15 mg Emulsion mixed type II collagen → preparation in 5 mg/ml with FCA

Mixed ① with ② by Syringe-syringe method. Day 0 Day 21

Anesthesia

Score 0 Score 1 Score 2 Score 3

WT (N=9)

Normal Swelling of 1 or Swelling, injections of Ankylosis Homo (N=9) more fingers All singers Hetero (N=11)

WT (non- WT CIA immunized) Immunohistochemistry

c c WT KO (Homo) c c JS JS

sy

sy

50μm 50μm A B CD3 (T cells) KO (Homo) CIA sy WT KO (Homo)

c

JS sy

50μm 50μm

C D WT CIA F4/80 (macrophages)

Immunohistochemistry Serum cytokines in CIA mice (1/4) WT KO (Homo)

50 μm 50μm 50μm CD79a (B-cells) WT KO (Homo)

WT *P<0.01 50μm 50μm Ly-6G (Neutrophils) KO

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Serum cytokines in CIA mice (2/4) Serum cytokines in CIA mice (3/4)

WT *P<0.01 WT *P<0.01

KO KO

Serum cytokines in CIA mice (4/4) IL-10 production in vitro

WT *P<0.01 a-CD3 (1 mg/ml) + a-CD28 ( 5 mg/ml)

KO

Summary of cytokine measurement Anti-type II collagen antibody in serum

WT > KO TNF-a, IL-6, IL-1b IFN-g IL-17 MIP‐1a

KO > WT IL-4 IL-10

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Proliferative response of LN cells Cell count of Spleen and lymph nodes

incorporation

TdR

H]

3 [

Proliferative response of splenic CD4+ T cells Bone marrow transfer for chimera

Donor Host

[WT→KO］

WT KO

Host Donor

[KO→WT]

KO WT

CIA on chimera mice Conclusions

• targeting of Cas-L/NEDD9

→ Reduction of CIA severity

→ Alteration of Cytokine balance

• Cas-L in hematopoietic cells may be more important for developing CIA.

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Contributors & Collaborators Future perspectives Institute of Medical Science The University of Tokyo Center for Experimental Research Hospital To elucidate Th17, Treg population in Cas-L -/- Medicine Division of Clinical Immunology mice Laboratory of Cell biology Tomoki Katayose Shinobu Saijo Yoshiko Kichikawa Shigeru Kakuta Hiromi Ichihara Yoichiro Iwakura Osamu Hosono Intracellular adaptor molecules: Hiroshi Kawasaki Graduate School of A therapeutic target for RA? Hirotoshi Tanaka Medicine Chikao Morimoto The University of Tokyo Sachiko Seo Mineo Kurokawa Hisamaru Hirai

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